Exploration And Study Of Clinical Scale Production Method Of Distal Respiratory Pluripotent Stem Cells

Objective:Respiratory diseases are a serious threat to human health,and the structural lung damage they cause has so far lacked effective clinical treatments.The first report of distal airway pluripotent stem cells(DASC),which can differentiate into a full spectrum of respiratory epithelial cells,and the method of culturing human DASC in large quantities in vitro in the journal Cell in 2011 opened up new prospects for stem cell therapy to repair and remodel airway and alveolar tissues.However,one of the important prerequisites for scaling up clinical trials and treatment of DASC is the development of more efficient,safe,and cost-effective methods for clinical-scale production of DASC.Methods:Bronchoscopic brushing of microscopic distal airway epithelial tissues from patients with respiratory diseases was used for DASC purification and amplification using a classical method based on the trophoblast layer of mouse embryonic fibroblasts(3T3-J2).High-throughput label-free microfluidic technology was developed to achieve efficient DASC-3T3-J2 separation during DASC passaging and amplification to replace classical methods such as differential digestion and immunomagnetic beads.We also explored the use of human amniotic mesenchymal stem cells(h AMSC)to replace 3T3-J2 trophoblast to achieve heterologous-free culture of human DASC;and the use of Collagen I and Laminin to replace trophoblast cells.Results:DASC with robust multispectral differentiation can be purified and cultured from the distal respiratory epithelial tissues of patients with respiratory diseases;novel microfluidic technology allows efficient cell separation based on the physical trait differences between DASC and 3T3-J2 trophoblast cells,achieving 80±5%DASC purity and 80±5 % recovery;AMSC can replace the classical 3T3-J2 trophoblast layer for long-term DASC culture;the use of Laminin and Collagen I pavement can replace the trophoblast layer for short-term DASC culture.Conclusions:This project proposes innovative and improved methods for the production of medical DASC.The use of microfluidic cell separation technology greatly enhances the feasibility and reduces the cost of clinical scale DASC production;the development of human-derived cell trophoblast and trophoblast-free culture methods can improve the safety of DASC production and reduce the cost of quality control.This study is of great value to promote the clinical trials and applications of DASC.